Articular cartilage lines the surfaces of joints and transmits the forces generated with loading. Due to limitations in cartilage's natural healing capacity, and given the increasing incidence of osteoarthritis, there exists a growing demand for cell-based strategies for repair. Tissue engineering, and particularly those approaches based on autologous mesenchymal stem cells (MSCs), is evolving as a clinically relevant technique to promote cartilage regeneration. Yet, the mechanics and ECM organization of engineered constructs for implantation have yet to match those of the native tissue and are insufficient to support joint loading. Most cartilage tissue engineering efforts replicate early stages of cartilage development, sequestering a high density of cartilaginous ECM producing cells in a defined volume. However, significant differences exist between these early rapid stages of cartilage formation and the gradual remodelling (maturation) that enables its adult function. With load-bearing use, cartilage ECM is wholly remodelled into the unique composition and architecture of the adult tissue. This transformative process is driven by a multitude of temporal factors (chemical, mechanical, and soluble). Our approach to cartilage tissue engineering adopts concepts of this complex developmental paradigm (from embryo to adult) with a synthetic hydrogel based on a natural ECM component, which provides initial receptor-matrix binding, controlled mechanics, and tailored degradation profiles. These gels, coupled with physiologic loading and temporal application of relevant soluble factors will re-create developmental microenvironments that both enable and encourage functional cartilage production by MSCs in 3-D culture. In the first Aim, MSCs will be encapsulated in HA hydrogels previously investigated as chondrocyte carriers with a range of structures that can influence cell viability, receptor-binding, diffusion, and ultimately, neocartilage properties and cultured under a range of conditions (i.e., cellular density and temporal variations in soluble factors). In the second Aim, novel HA hydrogels recently developed in our laboratory that degrade via both hydrolytic and enzymatic mechanisms will be investigated as 3D networks to promote MSC chondrogenesis and construct maturation. Temporal changes in degradation will be altered through the type of degradable group, crosslinking density, and copolymerization with HA macromers without hydrolytically degradable groups. In the third Aim, a mechanical loading bioreactor that applies sliding contact to hydrogel constructs will be applied to MSC-laden hydrogels identified in Aims 1 and 2, recapitulating normal physiologic loading patterns of cartilage. Mechanical preconditioning parameters will be evaluated in the short term (gene expression) and long term (matrix production, organization, and mechanics). These Aims were designed to allow the testing of our hypotheses that control over the MSC microenvironment and inclusion of signals present during normal development that are both permissive and instructive for cartilage formation and maturation will lead to final constructs with properties akin to native tissue. PUBLIC HEALTH RELEVANCE: This project develops a clinically-relevant approach to cartilage repair using MSC-laden hydrogels with temporally controlled structures subjected to physiologic mechanical conditioning to mimic the developmental paradigm of tissue formation and maturation that occurs from the embryo to adulthood. This biomaterial system provides receptor-matrix interactions, controlled mechanics, and tailored degradation profiles to enhance the differentiation of encapsulated MSCs and the accumulation and distribution of formed extracellular matrix (ECM). If successful, this approach would surmount a major hurdle in cartilage tissue engineering to provide a more structurally relevant ECM with enhanced mechanical properties to support the intense loads found in the joint. This cartilage tissue engineering technique could aid in the treatment of millions of patients afflicted with debilitating cartilage loss and degeneration due to trauma or disease.